Single-cell gel electrophoresis, also known as a “comet assay”, is a process by which DNA damage is quantified in individual cells. The process may be run under neutral conditions where strand breaks in DNA are identified or under alkaline conditions where alkali-labile sites or certain nucleobase modifications are identified. The principle of single-cell gel electrophoresis is that strand breaks lead to relaxation and unwinding of DNA under alkaline conditions. By subsequently applying an electric field, the DNA undergoes electrophoresis, moving under the influence of the electric field and forming a “comet” shape when viewed under a microscope. The amount of DNA in the comet “tail” relative to the amount remaining in the comet “head” is proportional to the number of strand breaks present. This ratio provides a quantified measure of the DNA damage present in a cell of interest.
Damage that can be detected by a comet assay include, for example, alkali labile sites, oxidatively generated nucleobase damage, double strand breaks, single strand breaks, and DNA cross-linking with other DNA or protein. The comet assay can also be used to measure DNA repair by noting the smaller size of the comet tail compared to damaged DNA after a given amount of time.
Single-cell gel electrophoresis is generally conducted in a series of steps (FIG. 1). A single cell of interest is first mixed with, and embedded in, agarose with a low melting point and then loaded onto a microscope slide, which has been pre-coated with an agarose gel matrix. After the gel matrix is allowed to set, it is then subjected to a number of pre-electrophoresis steps including lysing the cell at a high pH, washing, and optionally treating the cell with DNA repair enzymes, if needed. The presence of DNA strand breaks, and under high pH of the electrophoresis buffer, allows the cellular DNA to unwind. The slide is then transferred into an electrophoresis tank and electrophoresed. Then, a number of post-electrophoresis steps are performed including, for example, draining, neutralization, washing, staining, and drying. Finally, the cells are subjected to imaging and/or scoring for further analysis.
Although the comet assay is gaining popularity as industry and academic institutions began to adopt it for single-cell analysis and genotoxicology testing, some factors still limit its implementation. These factors include, for example, low sample throughput, inaccurate temperature control, and long sample workup procedure. Additionally, the numerous pre- and post-electrophoresis steps complicate the manipulation of multiple slides. In currently available methods, slides must be handled individually through each step, making the process laborious and time-consuming. Moreover, lengthy manipulation of slides presents an increased risk that the gels may become damaged, contaminated, or lost.
Methods presented previously in publications such as, for example, WO 2015025123, have improved comet assay efficiency by increasing the number of slides that can be accommodated during the assay process. However, there still remains a need of streamlining the assay such that the extent of slide manipulation can be reduced and the potential for automating the process can be improved.
In the past, the focus on increasing the rate of processing throughput had been placed on automating the scoring process, as it has been considered a bottleneck to the overall processing efficiency. However, with the automated scoring being improved recently, attention has been focused on streamlining other aspects of the comet assay to improve its throughput, particularly in slide manipulation and process automation.